Cyclohexylcarbamic Acid 3'- Or 4'-Substituted

4998 J. Med. Chem. 2004, 47, 4998-5008

Cyclohexylcarbamic Acid 3′-or4′-Substituted Biphenyl-3-yl Esters as Fatty Acid Amide Hydrolase Inhibitors: Synthesis, Quantitative Structure-Activity Relationships, and Molecular Modeling Studies

Marco Mor,† Silvia Rivara,† Alessio Lodola,† Pier Vincenzo Plazzi,† Giorgio Tarzia,‡ Andrea Duranti,*,‡ Andrea Tontini,‡ Giovanni Piersanti,‡ Satish Kathuria,§ and Daniele Piomelli§ Dipartimento Farmaceutico, Universita` degli Studi di Parma, Parco Area delle Scienze 27/A, I-43100 Parma, Italy, Istituto di Chimica Farmaceutica e Tossicologica, Universita` degli Studi di Urbino “Carlo Bo”, Piazza del Rinascimento 6, I-61029 Urbino, Italy, and Department of Pharmacology, University of California, Irvine, 360 MSRII, Irvine, California 92697-4625

Received December 15, 2003

Fatty acid amide hydrolase (FAAH) is a promising target for modulating endocannabinoid and fatty acid ethanolamide signaling, which may have important therapeutic potential. We recently described a new class of O-arylcarbamate inhibitors of FAAH, including the cyclohexylcarbamic acid biphenyl-3-yl ester URB524 (half-maximal inhibitory concentration, IC50 ) 63 nM), which have significant anxiolytic-like properties in rats. In the present study, by introducing a selected group of substituents at the meta and para positions of the distal phenyl ring of URB524, we have characterized structure-activity profiles for this series of compounds and shown that introduction of small polar groups in the meta position greatly improves inhibitory potency. Most potent in the series was the m-carbamoyl derivative URB597 (4i,IC50 ) 4.6 nM). Furthermore, quantitative structure-activity relationship (QSAR) analysis of an extended set of meta-substituted derivatives revealed a negative correlation between potency and lipophilicity and suggested that small-sized substituents may undertake polar interactions with the binding pocket of the enzyme. Docking studies and molecular dynamics simulations, using the crystal structure of FAAH, indicated that the O-biphenyl scaffold of the carbamate inhibitors can be accommodated within a lipophilic region of the substrate-binding site, where their folded shape mimics the initial 10-12 carbon atoms of the arachidonyl moiety of anandamide (a natural FAAH substrate) and methyl arachidonyl fluorophosphonate (a nonselective FAAH inhibitor). Moreover, substituents at the meta position of the distal phenyl ring can form hydrogen bonds with atoms located on the polar section of a narrow channel pointing toward the membrane- associated side of the enzyme. The structure-activity characterization reported here should help optimize the pharmacodynamic and pharmacokinetic properties of this class of compounds.

Introduction Fatty acid amide hydrolases (FAAHs)1,2 are intracellular enzymes responsible for the hydrolysis of endogenous fatty acid ethanolamides (FAEs),3,4 a reaction that, along with transport into cells,5-7 terminates the biological effects of these lipid mediators. At least two distinct classes of cellular receptors are thought to Figure 1. Chemical structures of anandamide (1) and URB524 mediate such effects. The polyunsaturated FAE anan- (2). damide8 (arachidonoylethanolamide, 1, Figure 1) activates cannabinoid receptors, G protein-coupled receptors Owing to the emerging physiological functions of the found in brain and immune cells that may play essential FAEs, small-molecule inhibitors that selectively target roles in the intrinsic regulation of pain, anxiety, and intracellular FAAH activity may not only be useful as 1 memory. On the other hand, the monounsaturated FAE research tools but also serve as prototypes for the 9,10 9 oleoylethanolamide (OEA, 18:1∆ ) activates the per- development of novel therapeutic agents.13-18 Therefore, R R oxisome proliferator-activated receptor- (PPAR- ), a starting from the assumption that carbamic acid esters nuclear receptor involved in the control of satiety, may act as site-directed inhibitors of FAAH, we have energy metabolism, and inflammation. The saturated developed a series of O-aryl-N-alkylcarbamic acid esters, FAE palmitoylethanolamide11,12 (PEA, 16:0) also is which are highly potent at inhibiting FAAH activity biologically active, but its mechanism of action is still both in vitro and in vivo, but do not significantly interact undefined. with several other serine hydrolases (e.g. acetylcho- * Corresponding author. Phone: (0039) 0722-330323. Fax: (0039) linesterase and monoglyceride lipase) or with cannab- 0722-2737. E-mail: [email protected]. inoid receptors. In rats, these compounds display pro- † Universita` degli Studi di Parma. ‡ Universita` degli Studi di Urbino “Carlo Bo”. found anxiolytic-like properties, which may be attributed § University of California, Irvine. to their ability to elevate brain anandamide levels.19

An initial structure-activity relationship (SAR) in- Scheme 1a vestigation into the steric requirements for the lipophilic O-aryl moiety of these compounds suggested that non- linearly shaped structures may be associated with greater inhibitory potencies.20 This idea is supported by two findings. First, the curved shape of the most successful FAAH inhibitors resembles the folded conformation assumed by fatty acids in complex with binding proteins, as well as one of the conformations predicted for anandamide binding to the CB1 cannabinoid receptor.21,22 Second, and more importantly, the crystal structure of FAAH inhibited by methyl arachidonyl fluorophosphonate (MAPF) revealed that the arachidonyl chain of this covalent inhibitor assumes a folded conformation in the substrate-binding site of the enzyme.23 Previous three-dimensional quantitative SAR (3D- QSAR) analyses of the alkylcarbamic acid aryl esters20 indicated that space occupancy of a region corresponding to the meta position of an O-phenyl ring is positively correlated with FAAH inhibition, which is in turn a Reagents and conditions: (a) c-C6H11NCO, Et3N, toluene, suggestive of a favorable interaction of the inhibitor with reflux, 1-32 h. the enzyme binding site. The most potent compound in this series was the biphenyl-3-yl derivative URB524 (2, Scheme 2a Figure 1), which inhibited FAAH activity in rat brain membranes with a half-maximal concentration (IC50) value of 63 nM. These experiments did not attempt, however, to identify the lipophilic and electronic, but only the steric, requirements of the binding site. There- fore, in the present study we have taken URB524 as the starting point for a systematic exploration of the effect of phenyl substitution on FAAH inhibition, introducing at the meta and para positions of the distal phenyl ring of URB524 a set of substituents with balanced variation in their lipophilic and electronic properties. The distal ring has been selected for two reasons. First, our previous 3D-QSAR analyses suggested that this moiety is critical to achieve significant inhibitory potency. Second, substitutions on the distal ring are in principle devoid of direct resonance effects upon the carbamate group, and are expected therefore a to yield compounds the potencies of which should Reagents and conditions: (a) Pd(PPh3)4, toluene, Na2CO3/H2O, - correlate directly with noncovalent binding interactions. EtOH, reflux, 1 3 h; (b) reaction conditions as in (a); (c) HI, reflux, 2-7.5 h for 3a,b,d,e,g,h,j,k,m,n or BBr ,25°C,1-18 h for 3c,o,q. Our experimental design comprised two steps. We 3 first tested the sensitivity of the meta and para positions tribromide (3c,o,q), hydrolysis of opportune 3-methoxy- of the distal phenyl ring to the lipophilic and electronic benzenes. The latter compounds were prepared by properties of a small set of moderate-size substituents. Suzuki coupling of arylboronic acid 5a and a haloben- Next, after the more responsive position of the phenyl - 26 27 ring was identified, we expanded the series of substit- zene (6a e,g,h,j,k,m, n,o, q) (Scheme 2). 6i was uents while maintaining significant and independent prepared from 3-bromobenzonitrile, employing sodium 28 - variation among the variables describing their lipo- perborate. Compounds 3i,r w were directly synthe- philic, electronic, and steric properties, as required to sized from the commercially available 3-hydroxyphen- investigate QSARs by multiple regression analysis ylboronic acid (5b) (Scheme 2). (MRA).24,25 The synthesis of 3p is reported in Scheme 4: the propenyl derivative 11, obtained by a Wittig reaction Chemistry from aldehyde 9 was reduced to give 8p, which was Cyclohexylcarbamic acid aryl esters 4a-c,e-i,l,k, eventually hydrolyzed to furnish the desired biphenol. m-w and 4x,y were obtained by addition of cyclohexyl- - - - isocyanate to phenylphenols 3a c,e,g i,k,m w Results and Discussion (Scheme 1), or 3x,y (Scheme 3), respectively. Removal of the protective benzyloxycarbonyl from 4x,y afforded We measured FAAH activity in rat brain membranes, 3 4j,d (Scheme 3). 4z was prepared by hydrogenation of using [ H]anandamide as a substrate. The IC50 values 4t. for compounds 4a-z are reported in Table 1. At 30- Compounds 3a-e,g,h,j,k,m-o,q were obtained by 100 µM, the compounds (i) had no effect on acetylcho- 57% hydriodic acid (3a,b,d,e,g,h,j,k,m,n), or boron linesterase activity (electric eel) or butyrylcholinesterase 5000 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 21 Mor et al.

a Scheme 3 Table 1. Inhibitory Potencies (IC50) of Tested Compounds on FAAH Activity

1 2 compd R R IC50 (nM) ( SEM URB524 H H 63 ( 9 4a HCF3 1587 ( 148 4b HCH3 155.4 ( 31.0 4c H C(O)NH2 5909 ( 951 4d HNH2 360 ( 59 4e H F 64.95 ( 14.00 4f H C(O)NHC(O)NH- 3017 ( 688 c-C6H11 ( a Reagents and conditions: (a) BzOC(O)Cl, NaHCO /H O, 20 4g CF3 H 145.7 16.0 3 2 ( h; (b) c-C H NCO, Et N, toluene, reflux, 8-32 h; (c) H , 10% 4h CH3 H 61.75 19.00 6 11 3 2 ( Pd/C, 3 atm, THF, MeOH, 65 °C, 24 h. 4i C(O)NH2 H 4.6 1.6 4j NH2 H 64.6 ( 9.0 ( Scheme 4a 4k F H 96.6 4.0 4l OC(O)NH-c-C6H11 H 361 ( 137 4m C6H5O H 420 ( 86 4n C6H5 H 565 ( 42 4o CH2C6H5 H 1857 ( 57 4p n-C3H7 H 110 ( 16 4q NO2 H 49.6 ( 2.0 4r SO2NH2 H 26.5 ( 4.5 4s C(O)CH3 H 9.1 ( 0.5 4t CN H 33.9 ( 7.0 4u OH H 8.65 ( 0.10 4v CH2OH H 8.67 ( 0.90 4w (CH2)2OH H 18.7 ( 4.5 4z CH2NH2 H 21177 ( 7277

of IC50 ratio of compounds 4b,4a and 4h,4g, respectively. Given the results of this first set of derivatives, further modifications at the 4′-position were discharged. a Reagents and conditions: (a) 2 M n-BuLi in hexane, N2,0 Moreover, compounds 4f and 4l demonstrated that the - °C, 2 h; (b) 78 to 25 °C, reflux, 20 h; (c) H2, PdO2,3atm,3h;(d) introduction of more extended fragments is detrimental HI, reflux, 3 h. to activity. activity (horse serum); (ii) did not displace the binding In order to search for statistical relationships between 3 of [ H]WIN-55212-2 (10 nM) to rat CB1 cannabinoid physicochemical properties and inhibitor potency, we receptors or human recombinant CB2 receptors; and (iii) inserted at the meta position twelve additional substit- did not affect [3H]anandamide (100 nM) transport in uents (4m-z, Table 1), which adequately span the space human astrocytoma cells in culture (data not shown). of lipophilic, steric, and electronic properties. Certain We first examined a small set of URB524 analogues substituents were also selected for their topological (4a-l) the substituents of which (methyl, trifluorometh- similarity with the carbamoyl group (4r) or with por- - yl, amino, and carbamoyl) represent the four combinations of it (4s, 4v,or4z). The IC50 values for 4m z - tions of positive and negative levels for the π and σ (σm indicate that hydrophilic (4q w) groups have a favor- or σp) descriptors; small (fluoro) and bulky (cyclohexyl- able impact on pharmacological activity. On the con- carbamoyloxy or cyclohexylureidocarbonyl) substituents trary, the introduction of large lipophilic groups (4m- were also included in this explorative set. The results p) is detrimental to activity. Several compounds in this suggested that substitutions in the meta position of the set are more active than URB524, although none of distal phenyl ring yield the most potent inhibitors. Thus, them is better than the m-carbamoyl derivative URB597 the 3′-methyl (4h) and 3′-amino (4j) derivatives were (4i). as potent as the parent compound (URB524), while the The nineteen (including hydrogen) biphenyl substit- 3′-carbamoyl derivative 4i (URB597) was an order of uents listed in Table 1 constitute a set with a large magnitude more potent than URB524. By contrast, with variation in both lipophilicity (almost 4 π units) and the only exception of the 4′-fluoro derivative 4e, all para- steric bulk (35 MR units). Furthermore, π and molar substituted compounds were less active than URB524. refractivity (MR) values are practically uncorrelated to In contrast with the general SAR trend, according to the electronic effects (r with σm of -0.19 and -0.16, which meta-substituted compounds were more active respectively), though some correlation is present be- than the corresponding para isomers, 3′-fluoro deriva- tween lipophilic (π) and steric (MR) descriptors (r ) tive 4k, the meta isomer of 4e, was less active than 0.63), due to the known difficulty in obtaining big URB524. The requirements of the group matching the hydrophilic substituents. area of the receptor near the 4′-position seem more strict Multiple regression analysis (MRA) employing eight than those of the 3′-group, as hinted by the comparison common physicochemical descriptors (π, σm, F, R, MR, Fatty Acid Amide Hydrolase Inhibitors Journal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5001

Table 2. QSAR Analysis: Correlation Matrix for the Nineteen Meta Substituents of Compounds URB524 and 4g-z

MR πσm σp FRLB1 π 0.63 σm -0.16 -0.19 σp -0.02 -0.14 0.89 F -0.19 -0.15 0.87 0.60 R 0.05 -0.12 0.60 0.89 0.19 L 0.86 0.42 -0.06 0.13 -0.15 0.23 B1 0.12 -0.07 0.49 0.59 0.29 0.56 0.30 B5 0.94 0.52 -0.14 -0.02 -0.18 0.06 0.75 0.11

2 L, B1, B5, see the correlation matrix in Table 2) and π did not yield a statistically significant model. However, a simple plot of pIC50 vs lipophilicity (not shown) indicated a clear relationship, albeit masked by the presence of an outlier, the aminomethyl derivative 4z. This can be attributed to the basicity of the compound, which is expected to be largely protonated at neutral Figure 2. Surface of the catalytic channel inside FAAH, pH. When 4z was omitted from the regression set, the colored by a lipophilicity scale going from high lipophilicity relationship between lipophilicity and potency gave the (brown) to high hydrophilicity (blue). The channel has an outlet regression equation 1. pointing to the membrane (bottom) and a funnel with a large opening toward the cytosol (high portion of figure, back). )- ( + ( Left: whole surface of the channel with catalytic Ser241 pIC50 0.49( 0.07)π 7.26( 0.09) (1) displayed. Right: vertical section of the channel (the frontal has been erased, so as to display the docked structure of 2 n ) 18 (URB524, 4g-w) r ) 0.74 s ) 0.37 URB524).

F ) 46.0 q2 ) 0.66 SDEP ) 0.40 Table 3. QSAR Analysis: Substituent Variables Employed in Eqs 1 and 2 and Observed pIC50 Values for Meta-Substituted Derivatives of 2 and Those Calculated by Eq 2 The negative correlation with lipophilicity was unex- pected in that we had previously hypothesized that the biphenyl moiety may mimic the fatty acyl chain of a FAE bound to the hydrophobic binding site of FAAH. Yet, no MRA model including up to five variables turned out to be statistically better than, or equivalent to, that represented in eq 1. Only the inclusion of an indicator pIC50 variable, set to one for substituents able to give hydro- compd R π MR HB obsd calcd gen bonds (HB), and to zero in other cases, allowed the URB524 H 0.00 1.03 0 7.20 7.24 detection of an alternative model (eq 2) having compa- 4g CF3 0.88 5.02 0 6.84 7.06 2 2 rable descriptive (r ) and predictive (q ) power. The 4h CH3 0.56 5.65 0 7.21 7.03 4i C(O)NH -1.49 9.81 1 8.34 7.64 pIC50 values calculated by eq 2 are reported in Table 3, 2 4j NH2 -1.23 5.42 1 7.19 7.84 together with independent variables employed in the 4k F 0.14 0.92 0 7.02 7.25 QSAR models. 4l OC(O)NH-c-C6H11 1.06 36.13 1 6.44 6.43 4m C6H5O 2.08 27.68 1 6.38 6.82 )- ( + ( + 4n C H 1.96 25.36 0 6.25 6.12 pIC50 0.046( 0.009) MR 0.80( 0.18) HB 6 5 ( 4o CH2C6H5 2.01 30.01 0 5.73 5.91 7.29( 0.17) 4p n-C3H7 1.55 14.96 0 6.96 6.60 4q NO2 -0.28 7.36 1 7.30 7.75 2 - n ) 18 (URB524, 4g-w) r ) 0.76 s ) 0.37 4r SO2NH2 1.82 12.28 1 7.58 7.53 4s C(O)CH3 -0.55 11.18 1 8.04 7.58 4t CN -0.57 6.33 1 7.47 7.80 2 F ) 23.2 q ) 0.68 SDEP ) 0.39 (2) 4u OH -0.67 2.85 1 8.06 7.96 4v CH2OH -1.03 7.19 1 8.06 7.76 - Although this model is more complex than that 4w (CH2)2OH 0.77 11.8 1 7.73 7.55 represented by eq 1, it could provide a possible inter- pretation for the positive effect of substituent hydro- indications reported above, even if none of them resulted philicity within a supposedly lipophilic binding pocket: more potent than the carbamoyl derivative 4i. eq 2 relates this behavior to the formation of hydrogen The resolution of the crystal structure of FAAH bonds between meta substituents and polar amino acid covalently bound to methyl arachidonyl phosphonate residues of the enzyme. This kind of interaction is (MAP)23 allowed us to rationalize our QSAR models by strictly dependent on distances and angles between the performing docking, and molecular dynamics (MD) atoms involved, which may explain the fact that, in the simulations, experiments. Bracey et al. observed that case of para-substituted compounds, no positive effect the binding site for MAP lies in a channel that spans for the polar carbamoyl (4c) and amino (4d) substituents the entire length of the enzyme, from the membrane- was observed. The last compounds of the series (4r- bound surface (Figure 2A, bottom) to the cytosol at the w), prepared to validate the temporary QSAR models opposite side (Figure 2A, top). The surface of this built with the data previously available, confirmed the channel, colored according to lipophilicity, is represented 5002 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 21 Mor et al.

Figure 3. Superposition of the biphenyl fragment of URB524 Figure 4. Docking of URB597 into the FAAH binding site. (white carbons) to the first two double bonds of the MAP Carbons of the inhibitor are colored in white, those of FAAH arachidonyl chain (yellow carbons), in the conformation ob- side chains in yellow, and those of the FAAH backbone in 23 served in the crystallized complex with FAAH. orange; other atoms are colored following the usual atom code. The hydrogen bonds of the carbamoyl group of URB597 with in Figure 2. The channel has a complex topography: it the enzyme are evidenced. The surface of the enzyme channel comprises a central hydrophilic area around the cata- has been left-rotated approximately 90° with respect to that lytic residue Ser241, which is surrounded in turn by represented in Figure 2. extended lipophilic surfaces. Proceeding toward the membrane, the duct bifurcates into a lipophilic bulge Phe381, Phe432, and Leu404 that only a hydrogen or (lower part of Figure 2A, back), in which the terminal fluorine atom may avoid clashes with their aryl or alkyl atoms of the arachidonyl chain of MAP are located, and groups (data not shown). a narrow tunnel with a hydrophilic ridge (lower part of The fact that URB597 is the most potent inhibitor of Figure 2A, front). The polar head of the FAEs might our series prompted us to investigate in greater detail temporarily link to this ridge en route to the catalytic its interaction with FAAH using theoretical methods. site. Docking analysis revealed that a binding mode alterna- After deletion of the MAP fragment from the enzyme tive to the one described above might be possible for this and refinement of the protein model (see Experimental compound. In fact, a 180° rotation of the molecule (i) Section), we performed molecular docking with URB524 brings the N-cyclohexyl ring in a portion of the space and URB597 (4i). The results of these analyses showed occupied by the arachidonyl chain of MAP; and (ii) that the biphenyl moiety of URB524 may readily replace allows the biphenyl scaffold to lie in the funnel pointing the arachidonyl chain of MAP, with the meta position to the cytosolic outlet, which is part of the FAAH of the distal phenyl ring pointing exactly toward the channel. In this orientation it is still possible for the hydrophilic ridge (Figure 2B). Indeed, a superposition carbamoyl group of URB597 to form two hydrogen placing the first two double bonds of the arachidonyl bonds, one with the backbone carbonyl of Leu380 and chain of MAP, in the conformation of its complex with the other with the side-chain NH2 of Gln273 (data not FAAH, onto the biphenyl moiety of URB524 highlights shown). the steric similarity of the two fragments and supports To test which of the two orientations is more stable our initial hypothesis (Figure 3). we performed MD runs, maintaining the tertiary struc- Docking of URB597, the most active compound of the ture of FAAH observed in its crystallized complex with present series, suggests that its carbamoyl group may MAP and allowing movement of amino acid residues form two distinct hydrogen bonds: one as an acceptor, located at a maximum distance of 8 Å from the docked with the hydroxyl group of Thr488, and the other as a carbamate inhibitor. Starting from the orientation of donor, with the main chain carbonyl group of Leu192 Figure 4, URB597 gave a stable oscillation around a (Figure 4). Docking of other meta-substituted structures, conformation very similar to its initial one, with an with the common biphenyl scaffold held in the same average distance between the nucleophilic oxygen of lipophilic region as in Figure 4, showed that all polar Ser241 and the electrophilic carbamate carbon of 3.00 groups can form hydrogen bonds with the enzyme. ( 0.15 Å (mean ( SD). On the other hand, when MD However, the hydrogen bond of the carbamoyl group of runs were started from the alternative orientation, the URB597 is the strongest, possibly because its bidentate ligand position was much less stable, the average structure is absent in the acetyl and hydroxymethyl distance between the two centers being of 4.10 ( 0.65 derivatives 4s and 4v. Accordingly, URB597 is more Å. Moreover, the m-carbamoyl group of URB597 left its potent than the latter compounds (Table 1). This original position and was able to undergo alternative structure-based analysis, evidencing a role for specific polar interactions only at the cost of a worse adaptation hydrogen bonding interactions of our inhibitors with of the carbamate fragment to the catalytic site. These FAAH, may account for the empirical correlation de- results suggest that the binding mode depicted in Figure scribed by eq 2 and provide an explanation for the 4 may favor the nucleophilic attack of Ser241 after negative correlation with lipophilicity (eq 1). The low substrate recognition. tolerance to para substitution could be explained by the The stabilizing effect of the 3′-carbamoyl group in this observation that, in the docking mode described above, binding orientation was further confirmed by comparing the para position resulted so close to the side chains of the MD trajectory of URB597 with that of its parent Fatty Acid Amide Hydrolase Inhibitors Journal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5003 compound URB524. The first structure showed reduced (0.280 g). Mp: 147-148 °C (EtOH). MS (EI): m/z 238 (M+), 1 - fluctuations, with respect to the initial conformation, 69 (100). H NMR (CDCl3): δ 1.17 2.07 (m, 10H), 3.59 (m, - - indicating that the additional interaction provided by 1H), 4.96 (d, 1H), 7.15 7.21 (m, 1H), 7.39 7.50 (m, 3H), 7.69 (s, 4H) ppm. IR (KBr): 3297, 1743, 1706 cm-1. Anal. (C H F - the carbamoyl group contributes to limit the movements 20 20 3 NO2)C,H,N. of the inhibitor within the active site; this increases the Cyclohexylcarbamic Acid 4′-Methylbiphenyl-3-yl Es- probability of a reaction between Ser241 and the car- ter (4b). White crystals were obtained. Yield: 98% (0.303 g). bamate group, and could be related to the observed Mp: 149-150 °C (MeOH). MS (EI): m/z 184, 61 (100). 1H NMR improvement of potency conferred by polar substituents (CDCl3): δ 1.17-2.07 (m, 10H), 2.40 (s, 3H), 3.59 (m, 1H), 4.94 in the 3′-position. (br d, 1H), 7.10 (m, 1H), 7.22-7.51 (m, 7H) ppm. IR (Nujol): -1 In conclusion, our findings indicate that the biphenyl 3300, 1736, 1698, 1605, 1535, 1459 cm . Anal. (C22H23NO2) C, H, N. scaffold in O-arylcarbamate inhibitors of FAAH may Cyclohexylcarbamic Acid 4′-Carbamoylbiphenyl-3-yl mimic the initial fatty acyl segment of anandamide and Ester (4c). White crystals were obtained. Yield: 10% (0.034 other FAEs, as they interact with the enzyme’s active g). Mp: 223-225 °C (EtOAc). MS (EI): m/z 213 (M+), 197 1 site. The results further suggest that polar substituents (100). H NMR (CDCl3): δ 1.20-2.00 (m, 10H), 3.50 (m, 1H), in the meta position of the distal phenyl group can form 5.00 (br d, 1H), 5.60 (br s, 1H), 6.10 (br s, 1H), 7.15 (m, 1H), hydrogen bonds with hydrophilic amino acid residues 7.40 (m, 3H), 7.65 (d, 2H), 7.90 (d, 2H) ppm. IR (KBr): 3379, -1 within the FAAH channel. MD simulations supported 3331, 2926, 2849, 1705, 1627, 1578 cm . Anal. (C20H22N2O3) C, H, N. this hypothesis, showing that such hydrogen bonding Cyclohexylcarbamic Acid 4′-Fluorobiphenyl-3-yl Ester may increase the time spent by the electrophilic car- (4e). White crystals were obtained. Yield: 80% (0.251 g). Mp: bamate carbon near the catalytic residue Ser241, which 131-133 °C (EtOH). MS (EI): m/z 313 (M+), 188 (100). 1H should in turn favor the catalytic process. Since car- NMR (CDCl3): δ 1.17-2.06 (m, 10H), 3.59 (br s, 1H), 4.97 (br bamate inhibitors of FAAH activity exert potent in vivo d, 1H), 7.08-7.58 (m, 8H) ppm. IR (Nujol): 3316, 3294, 3055, -1 effects of therapeutic relevance,29 including anxiolytic- 1747, 1709, 1587, 1579, 1534 cm . Anal. (C19H20FNO2)C,H, like actions, a detailed knowledge of their SAR profile N. ′ should be useful for further developments in this field. Cyclohexylcarbamic Acid 4 -(3-Cyclohexylureidocar- bonyl)biphenyl-3-yl Ester (4f). 4f was isolated as a side Experimental Section product of the synthesis of 4c: white crystals. Yield: 11% (0.051 g). Mp: 184-186 °C (EtOAc). MS (EI): m/z 213 (M+), (a) Chemicals, Materials, and Methods. All reagents 1 197 (100), 139, 115. H NMR [CD3C(O)CD3]: δ 1.10-2.00 (m, were purchased from Sigma-Aldrich in the highest quality 20H), 3.48 (m, 2H), 5.10 (br s, 1H), 6.70 (br s, 2H), 7.15 (m, commercially available. Solvents were RP grade unless oth- 1H), 7.50 (m, 3H), 7.75 (d, 2H), 8.04 (d, 2H) ppm. IR (KBr): erwise indicated. Purification of the crude material obtained 3340, 3334, 3178, 2936, 2851, 1706, 1627, 1235 cm-1. Anal. from the reactions, affording the desired products, was carried (C H N O )C,H,N. out by flash column chromatography on silica gel (Kieselgel 27 33 3 4 Cyclohexylcarbamic Acid 3′-Trifluoromethylbiphenyl- 60, 0.040-0.063 mm, Merck). TLC analyses were performed 3-yl Ester (4g). White crystals were obtained. Yield: 78% on precoated silica gel on aluminum sheets (Kieselgel 60 F , 254 (0.283 g). Mp: 102-104 °C (EtOH). MS (EI): m/z 363 (M+), Merck). Melting points were determined on a Bu¨ chi SMP-510 238 (100). 1H NMR (CDCl ): δ 1.17-2.07 (m, 10H), 3.60 (m, capillary melting point apparatus and are uncorrected. The 3 1H), 4.96 (br d, 1H), 7.18 (m, 1H), 7.37-7.82 (m, 7H) ppm. IR structures of the unknown compounds were unambiguously -1 1 (Nujol): 3298, 1742, 1709 cm . Anal. (C20H20F3NO2)C,H,N. assessed by MS, H NMR, IR, and elemental analysis. EI-MS ′ spectra (70 eV) were recorded with a Fisons Trio 1000 Cyclohexylcarbamic Acid 3 -Methylbiphenyl-3-yl Es- + ter (4h). White crystals were obtained. Yield: 78% (0.241 g). spectrometer. Only molecular ions (M ) and base peaks are + 1 1 Mp: 133 °C (MeOH). MS (EI): m/z 309 (M ), 184 (100). H given. H NMR spectra were recorded on a Bruker AC 200 - spectrometer. Chemical shifts (δ scale) are reported in parts NMR (CDCl3): δ 1.17 2.07 (m, 10H), 2.42 (s, 3H), 3.59 (m, 1H), 4.94 (br d, 1H), 7.08-7.40 (m, 8H) ppm. IR (Nujol): 3305, per million (ppm) relative to the central peak of the solvent. -1 IR spectra were obtained on a Shimadzu FT-8300 or a Nicolet 1741, 1703, 1600, 1578, 1535, 1459 cm . Anal. (C20H23NO2) Atavar 360 FT spectometer. Absorbances are reported in ν C, H, N. - ′ (cm 1). Elemental analyses were performed on a Carlo Erba Cyclohexylcarbamic Acid 3 -Carbamoylbiphenyl-3-yl analyzer. All products had satisfactory (within (0.4 of theo- Ester (4i). White crystals were obtained. Yield: 33% (0.112 retical values) C, H, N analysis results. g). Mp: 178 °C (EtOH) (sealed capillary tube). MS (EI): m/z 1 - General Procedure for the Synthesis of Cyclohexyl- 213 (100). H NMR (CDCl3): δ 1.17 1.43 (m, 6H), 1.76 (m, carbamic Acid Aryl Esters (4a-c,e-i,k,l,m-y). To a 2H), 2.04 (m, 2H), 3.57 (m, 1H), 4.97 (br d, 1H), 5.63 (br s, - stirred solution of the suitable aryl alcohol 3a-c,e,g-i,k,m-y 1H), 6.14 (br s, 1H), 7.16 (m, 1H), 7.39 7.56 (m, 4H), 7.77 (m, 2H), 8.03 (s, 1H) ppm. IR (CHCl ): 3301, 3142, 1693, 1666, (1 mmol) in toluene (6 mL) were added Et3N (0.006 g, 0.01 3 1627, 1604, 1573 cm-1. Anal. (C H N O )C,H,N. mL, 0.06 mmol) and c-C6H11NCO (138 mg, 0.14 mL, 1.1 mmol). 20 22 2 3 After the reactants were refluxed for5h(4r,1h;4q,8h; Cyclohexylcarbamic Acid 3′-Fluorobiphenyl-3-yl Ester 4f,i,l,o,p,u, 18 h), a further amount of c-C6H11NCO [0.046 g, (4k). White crystals were obtained. Yield: 77% (0.241 g). Mp: + 0.05 mL, 0.37 mmol; 0.138 g, 0.14 mL, 1.1 mmol in the case of 128-130 °C (EtOH). MS (EI): m/z 313 (M ), 188 (100). 1H - 4e,g,o,y (no such further addition was necessary in the cases NMR (CDCl3): δ 1.17 2.06 (m, 20H), 3.59 (m, 1H), 4.96 (br d, of 4f,i,p,q,r,v,w)] was added, and the mixture was again 1H), 6.99-7.44 (m, 8H) ppm. IR (Nujol): 3309, 3294, 1741, -1 reacted for 3 h. A third amount of c-C6H11NCO (0.091 g, 0.09 1709, 1580, 1539 cm . Anal. (C19H20FNO2)C,H,N. mL, 0.726 mmol) was needed in the case of 4g,x, the synthesis Cyclohexylcarbamic Acid 3′-Cyclohexylcarbamoyl- of which overall required 32 h. The mixture was then cooled oxybiphenyl-3-yl Ester (4l). Isolated from the crude of and concentrated. Purification of the residue by column synthesis of 4u: white crystals. Yield: 20% (0.090 g). Mp: chromatography (cyclohexane/EtOAc 85:15 for 4b,g,h,k,o; 4:6 214-216 °C (EtOH). MS (EI): m/z 186 (M+), 157 (100). 1H for 4i; 1:1 for 4f,p; 55:45 for 4r; 65:35 for 4l,u; 7:3 for 4q,v; NMR (CDCl3): δ 1.22-2.06 (m, 20H), 3.60 (m, 2H), 4.93 (br d, - 75:25 for 4s,x; 8:2 for 4a,e,t; 9:1 for 4m,p;CH2Cl2 for 4n;CH2- 2H), 7.10 (m, 2H), 7.30 7.50 (m, 6H) ppm. IR (KBr): 3423, -1 ‚ Cl2/EtOAc 100:1 for 4y; toluene/MeOH 95:5 and cyclohexane/ 3312, 1765, 1644 cm . Anal. (C26H32N2O4S 0.1c-C6H11)C,H, EtOAc 7:3 for 4w) and recrystallization gave 4a-c,e-i,k,l,m- N. w. Cyclohexylcarbamic Acid 3′-Phenoxybiphenyl-3-yl Es- Cyclohexylcarbamic Acid 4′-Trifluoromethylbiphenyl- ter (4m). White crystals were obtained. Yield: 74% (0.287 g). 3-yl Ester (4a). Colorless crystals were obtained. Yield: 77% Mp: 142 °C (EtOH). MS (EI): m/z 262, 51 (100). 1H NMR 5004 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 21 Mor et al.

(CDCl3): δ 1.17-2.06 (m, 10H), 3.58 (m, 1H), 4.93 (br d, 1H), Cyclohexylcarbamic Acid 3′-Benzyloxycarbonylami- 6.96-7.44 (m, 13H) ppm. IR (Nujol): 3305, 1741, 1709, 1578, nobiphenyl-3-yl Ester (4x). A vitreous solid was obtained. -1 + 1 1540 cm . Anal. (C25H25NO3)C,H,N. Yield: 86% (0.382 g). MS (EI): m/z 319 (M ), 91 (100). H NMR [1,1′;3′,1′′]-Terphenyl-3-yl Carbamic Acid Cyclohexyl (CDCl3): δ 1.16-2.06 (m, 10H), 3.58 (m, 1H), 4.97 (br d, 1H), Ester (4n). White crystals were obtained. Yield: 74% (0.275 5.23 (s, 2H), 6.80 (br s, 1H), 7.12 (m, 1H), 7.30-7.41 (m, 11H), g). Mp: 177-179 °C (EtOAc). MS (EI): m/z 371 (M+), 246 7.61 (s, 1H) ppm. IR (Nujol): 3308, 1709, 1606, 1537 cm-1. 1 (100). H NMR (CDCl3): δ 1.17-2.06 (m, 10H), 3.60 (m, 1H), Cyclohexylcarbamic Acid 4′-Benzyloxycarbonylami- 4.95 (br d, 1H), 7.12-7.80 (m, 13H) ppm. IR (KBr): 3301, 2923, nobiphenyl-3-yl Ester (4y). A white solid was obtained. -1 2853, 1744, 1707 cm . Anal. (C25H25NO2)C,H,N. Yield: 94% (0.418 g). Mp: 195-196 °C (EtOH). MS (EI): m/z + 1 Cyclohexylcarbamic Acid 3′-Benzylbiphenyl-3-yl Es- 319 (M ), 167. H NMR (CDCl3): δ 1.16-2.18 (m, 10H), 3.59 ter (4o). White crystals were obtained. Yield: 70% (0.270 g). (m, 1H), 4.94 (br d, 1H), 5.23 (s, 2H), 6.74 (br s, 1H), 7.09 (m, Mp: 112-114 °C (EtOH). MS (EI): m/z 260 (M+), 165 (100). 1H), 7.32-7.56 (m, 12H) ppm. IR (Nujol): 3332, 1703, 1600, 1 -1 H NMR (CDCl3): δ 1.20-1.50 (m, 6H), 1.60 (m, 2H), 2.00 (m, 1540 cm . 2H), 3.58 (m, 1H), 4.04 (s, 2H), 4.93 (br d, 1H), 7.10-7.50 (m, General Procedure for the Synthesis of 3′-(or 4′-)- 13H) ppm. IR (KBr): 3154, 1733, 1602, 1558, 1471 cm-1. Anal. Substituted-3-methoxybiphenyls (8a-e,g,h,j,k,m-o,q,9) (C26H27NO2)C,H,N. and 3-Substituted-1-hydroxybiphenyls (3i,r-w). To a Cyclohexylcarbamic Acid 3′-Propylbiphenyl-3-yl Ester stirred solution of the opportune halobenzene 6a-e,g-k,m- (4p). Colorless crystals were obtained. Yield: 86% (0.290 g). o,q-w,7 (15.5 mmol) in toluene (100 mL) were added Pd- + Mp: 89-90 °C (petroleum ether). MS (EI): m/z 337 (M ), 212 (PPh3)4 (0.726 g, 0.63 mmol), a solution of Na2CO3 (10.323 g, 1 (100). H NMR (CDCl3): δ 0.97 (t, 3H), 1.16-1.79 (m, 10H), 97.37 mmol) in H2O (50 mL), and a solution of the suitable 2.04 (m, 2H), 2.65 (t, 2H), 3.59 (m, 1H), 4.94 (br d, 1H), 7.08- 3-methoxyphenyl boronic acid (5a) (31 mmol) or 3-hydroxy- 7.43 (m, 8H) ppm. IR (Nujol): 3310, 1736, 1703, 1546 cm-1. phenyl boronic acid (5b) (15.5 mmol) in EtOH (44 mL) under Anal. (C22H27NO2)C,H,N. N2 atmosphere. The mixture was vigorously stirred under Cyclohexylcarbamic Acid 3′-Nitrobiphenyl-3-yl Ester reflux for the opportune time (1 h for 3r-t,v,w,8b,h,j,9;2h - (4q). Colorless needles. Yield: 75% (0.255 g). Mp: 138-142 for 3i,8a,m,q;3hfor3u,8c e,g,k,n,o) and cooled, H2O was + °C (EtOH). MS (EI): m/z 215 (M ), 141 (100). 1H NMR added, and the mixture was extracted with CH2Cl2 (EtOAc in (CDCl3): δ 1.17-2.07 (m, 10H), 3.59 (m, 1H), 4.98 (br d, 1H), the case of 3i,r,t,w,8d,m; the acidification of the aqueous 7.21 (m, 1H), 7.45 (m, 3H), 7.61 (t, 1H), 7.92 (d, 1H), 8.22 (m, phase with 2 N HCl was necessary for 3r). The combined 1H), 8.45 (m, 1H) ppm. IR (Nujol): 3289, 1744, 1701, 1530, organic layers were dried (Na2SO4) and concentrated. Purifica- -1 1344 cm . Anal. (C19H20N2O4)C,H,N. tion of the residue by column chromatography (cyclohexane/ Cyclohexylcarbamic Acid 3′-Sulfamoylbiphenyl-3-yl EtOAc 200:1 for 8m; 98:2 for 8b,h,n,o; 97:3 for 8e; 95:5 for Ester (4r). White crystals which decompose starting from 115 8g,k; 85:15 for 8q,9; 8:2 for 8a; 75:25 for 3s; 7:3 for 3u,8j; 65: °C (EtOAc/petroleum ether) were obtained. Yield: 26% (0.093 35 for 3t,v; 6:4 for 8d; 1:1 for 3r; 7:3 to 6:4 to 1:1 for 3w; 2:8 + 1 for 3i; EtOAc for 8c) gave 8a,d,e,g,h,j,k,m-o,9 and 3r,s,v,w g). MS (EI): m/z 249 (M ), 169 (100). H NMR [CD3C(O)CD3]: δ 1.16-2.05 (m, 10H), 3.48 (br s, 1H), 6.68 (s, 2H), 6.77 (br d, as oils and 8b,c,q and 3i,t,u as solids. 1H), 7.17 (d, 1H), 7.48 (m, 3H), 7.67 (t, 1H), 7.91 (d, 2H), 8.17 3-Methoxy-4′-trifluoromethylbiphenyl (8a).30 A colorless + (s, 1H) ppm. IR (KBr): 3347, 3310, 3262, 1698, 1540 cm-1. oil was obtained. Yield: 95% (3.714 g). MS (EI): m/z 252 (M , 1 30 Anal. (C19H22N2O4‚0.2C6H14)C,H,N. 100). The H NMR spectrum is according to literature. Cyclohexylcarbamic Acid 3′-Acetylbiphenyl-3-yl Ester 3-Methoxy-4′-methylbiphenyl (8b).31 White crystals were (4s). Pearly scales were obtained. Yield: 72% (0.234 g). Mp: obtained. Yield: 94% (2.889 g). Mp: 75-76 °C (MeOH). MS + + 1 130-132 °C (EtOH). MS (EI): m/z 212 (M ), 197 (100). 1H (EI): m/z 198 (M ), 69 (100). H NMR (CDCl3): δ 2.40 (s, 3H), NMR (CDCl ): δ 1.17-2.07 (m, 10H), 2.67 (s, 3H), 3.59 (m, 3.87 (s, 3H), 6.88 (m, 1H), 7.11-7.52 (m, 7H) ppm. IR (neat): 3 - 1H), 4.98 (br d, 1H), 7.17 (m, 1H), 7.39-7.58 (m, 4H), 7.79 (d, 3055, 3006, 1611, 1584, 1567, 1519, 1486 cm 1. 1H), 7.95 (d, 1H), 8.17 (s, 1H) ppm. IR (Nujol): 3305, 1736, 3′-Methoxybiphenyl-4-carboxylic Acid Amide (8c). White -1 - 1709, 1687, 1600, 1534 cm . Anal. (C21H23NO3)C,H,N. crystals were obtained. Yield: 55% (1.937 g). Mp: 198 200 + Cyclohexylcarbamic Acid 3′-Cyanobiphenyl-3-yl Ester °C (EtOH). MS (EI): m/z 227 (M ), 199 (100). 1H NMR (4t). White crystals were obtained. Yield: 80% (0.257 g). Mp: (CDCl3): δ 3.90 (s, 3H), 5.60 (br s, 1H), 6.10 (br s, 1H), 6.95 105-107 °C (EtOH) (sealed capillary tube). MS (EI): m/z 320 (dd, 1H), 7.17 (m, 2H), 7.37 (t, 1H), 7.67 (d, 2H), 7.89 (d, 2H) + 1 (M ), 195 (100). H NMR (CDCl3): δ 1.22-2.07 (m, 10H), 3.58 ppm. (m, 1H), 4.97 (br d, 1H), 7.18 (m, 1H), 7.36-7.86 (m, 7H) ppm. 3′-Methoxybiphenyl-4-yl Amine (8d).32 A yellow oil was -1 + IR (Nujol): 3305, 2235, 1741, 1703 cm . Anal. (C20H20N2O2) obtained. Yield: 57% (1.760 g). MS (EI): m/z 199 (M , 100), 1 C, H, N. 156. H NMR (CDCl3): δ 3.86 (s and br s, 5H), 6.74-7.44 (m, ′ 8H) ppm. IR (neat): 3457, 3370, 3218, 3033, 3001, 2952, 2936, Cyclohexylcarbamic Acid 3 -Hydroxybiphenyl-3-yl Es- - ter (4u). White crystals were obtained. Yield: 45% (0.148 g). 1600 cm 1. Mp: 68-70 °C (EtOAc/hexane). MS (EI): m/z 186 (M+), 157 4′-Fluoro-3-methoxybiphenyl (8e).33 A colorless oil was 1 + (100). H NMR (CDCl3): δ 1.20-1.50 (m, 6H), 1.70 (m, 2H), obtained. Yield: 62% (1.943 g). MS (EI): m/z 202 (M ), 133 2.00 (m, 2H), 3.55 (m, 1H), 4.95 (br d, 1H), 5.10 (br s, 1H), (100). 6.80 (m, 1H), 7.05 (m, 1H), 7.15 (m, 2H), 7.40 (m, 4H) ppm. IR 3-Methoxy-3′-trifluoromethylbiphenyl (8g). A colorless -1 + (KBr): 3390, 2927, 2852, 1708, 1650 cm . Anal. (C19H21NO3‚ oil was obtained. Yield: 93% (3.636 g). MS (EI): m/z 252 (M ), 1 0.2EtOAc) C, H, N. 69 (100). H NMR (CDCl3): δ 3.89 (s, 3H), 6.93-7.84 (m, 8H) Cyclohexylcarbamic Acid 3′-Hydroxymethylbiphenyl- ppm. IR (neat): 3071, 3001, 2952, 2838, 1600, 1578, 1486, 1464 - 3-yl Ester (4v). Colorless tablets were obtained. Yield: 64% cm 1. (0.208 g). Mp: 137-140 °C (EtOH). MS (EI): m/z 325 (M+), 3-Methoxy-3′-methylbiphenyl (8h).34 A pink oil was 1 + 153 (100). H NMR (CDCl3): δ 1.17-2.06 (m, 11H), 3.59 (m, obtained. Yield: 95% (2.919 g). MS (EI): m/z 198 (M ), 155 1H), 4.76 (s, 2H), 4.98 (br d, 1H), 7.13 (m, 1H), 7.34-7.54 (m, (100). 6H), 7.59 (s, 1H) ppm. IR (KBr): 3446, 3272, 3061, 1709, 1595, 3′-Methoxybiphenyl-3-yl Amine (8j). A yellow oil was -1 + 1540 cm . Anal. (C20H23NO3)C,H,N. obtained. Yield: 94% (2.903 g). MS (EI): m/z 199 (M , 100), 1 Cyclohexylcarbamic Acid 3′-(2-Hydroxyethyl)biphen- 156. H NMR (CDCl3): δ 3.87 (s and br s, 5H), 6.69 (s, 1H), yl-3-yl Ester (4w). White crystals were obtained. Yield: 13% 6.86-7.38 (m, 7H) ppm. IR (neat): 3457, 3365, 3213, 3033, (0.044 g). Mp: 106 °C (acetone/petroleum ether). MS (EI): m/z 2963, 2936, 1622, 1600, 1578 cm-1. 1 35 214, 82 (100). H NMR [CD3C(O)CD3]: δ 1.29-1.97 (m, 11H), 3′-Fluoro-3-methoxybiphenyl (8k). A pale yellow oil 2.88 (t, 2H), 3.47 (m, 1H), 3.80 (t, 2H), 6.73 (br s, 1H), 7.06- was obtained. Yield: 83% (2.602 g). MS (EI): m/z 202 (M+, 1 7.54 (m, 8H) ppm. IR (Nujol): 3457, 3305, 3256, 1712, 1709, 100). H NMR (CDCl3): δ 3.89 (s, 3H), 6.91-7.43 (m, 8H) ppm. -1 -1 1562, 1535 cm . Anal. (C21H25NO3)C,H,N. IR (neat): 3071, 2995, 2963, 2936, 2838, 1608, 1587 cm . Fatty Acid Amide Hydrolase Inhibitors Journal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5005

3-Methoxy-3′-phenoxybiphenyl (8m). A light yellow oil 4′-Trifluomethylbiphenyl-3-ol (3a). Colorless needles was obtained. Yield: 42% (1.800 g). MS (EI): m/z 276 (M+, were obtained. Yield: 70% (0.584 g). Mp: 73-75 °C (petroleum 1 + 1 100). H NMR (CDCl3): δ 3.87 (s, 3H), 6.89-7.45 (m, 13H) ether). MS (EI): m/z 238 (M ), 69 (100). H NMR (CDCl3): δ ppm. IR (neat): 3061, 3033, 2995, 2925, 2849, 2832, 1595, 1573 4.91 (s, 1H), 6.88 (m, 1H), 7.08 (m, 1H), 7.18 (d, 1H), 7.35 (t, cm-1. 1H), 7.69 (s, 4H) ppm. IR (Nujol): 3280, 1616, 1600, 1590, 1573 3-Methoxy-[1,1′;3,1′′]terphenyl (8n). An opaque white oil cm-1. was obtained. Yield: 97% (3.914 g). MS (EI): m/z 260 (M+), 4′-Methylbiphenyl-3-ol (3b). White crystals were ob- 1 77 (100). H NMR (CDCl3): δ 3.89 (s, 3H), 6.92-7.82 (m, 13H) tained. Yield: 85% (0.548 g). Mp: 74-75 °C (cyclohexane). MS -1 + 1 ppm. IR (film): 3061-2834 cm . (EI): m/z 184 (M ), 165 (100). H NMR (CDCl3): δ 2.40 (s, 3′-Benzyl-3-methoxybiphenyl (8o). An amber oil was 3H), 4.87 (s, 1H), 6.81 (m, 1H), 7.06 (t, 1H), 7.14-7.50 (m, 6H) obtained. Yield: 95% (4.040 g). MS (EI): m/z 274 (M+), 165 ppm. IR (Nujol): 3289, 1589, 1567, 1464 cm-1. 1 (100). H NMR (CDCl3): δ 3.87 (m, 12H), 6.90 (dd, 1H), 4.06 4′-Aminobiphenyl-3-ol (3d). A light-brown solid was (s, 2H), 3.87 (s, 3H) ppm. IR (Nujol): 3154, 2962, 1599, 1468, obtained. Yield: 77% (0.499 g). Mp: 163-165 °C (EtOAc/ 1381 cm-1. petroleum ether). MS (EI): m/z 185 (M+), 156 (100). 1H NMR 3′-Methoxy-3-nitrobiphenyl (8q). Gray sheaves were (CDCl3): δ 3.81 (br s, 2H), 6.63 (m, 3H), 6.89 (m, 2H), 7.09 (t, obtained. Yield: 93% (3.304 g). Mp: 75-76 °C (EtOH) [lit.: 1H), 7.26 (m, 2H), 8.57 (s, 1H) ppm. IR (Nujol): 3382, 3313, 77-78 °C (EtOH)].36 MS (EI): m/z 229 (M+, 100), 168. IR 1596, 1579 cm-1. (Nujol): 1605, 1584, 1567, 1524, 1345 cm-1. The 1H NMR 4′-Fluorobiphenyl-3-ol (3e).41 An amorphous solid was spectrum is according to literature.36 obtained. Yield: 53% (0.349 g). MS (EI) is according with 3′-Methoxybiphenyl-3-carbaldehyde (9). An opaque yel- literature.41 low oil was obtained. Yield: 82% (2.698 g). MS (EI): m/z 212 3′-Trifluomethylbiphenyl-3-ol (3g). A white solid was (M+), 168 (100). The 1H NMR and IR spectra are in agreement obtained. Yield: 82% (0.684 g). Mp: 63-66 °C (petroleum 37 + 1 with literature. ether). MS (EI): m/z 238 (M , 100). H NMR (CDCl3): δ 4.97 3′-Hydroxybiphenyl-3-carboxylic Acid Amide (3i). A (s, 1H), 6.88 (m, 1H), 7.08 (m, 1H), 7.16-7.82 (m, 6H) ppm. sand-colored solid was obtained. Yield: 98% (3.239 g). Mp: IR (Nujol): 3341, 1618, 1598, 1583 cm-1. 42 148-151 °C (after digestion with i-Pr2O). MS (EI): m/z 213 3′-Methylbiphenyl-3-ol (3h). A brown oil was obtained. + 1 + 1 (M , 100). H NMR [CDCl3/(CD3)2SO]: δ 6.06 (br s, 1H), 6.59 Yield: 90% (0.580 g). MS (EI): m/z 184 (M , 100). H NMR (m, 1H), 6.85 (m, 2H), 7.01 (t, 1H), 7.23 (t, 1H), 7.35 (br s, (CDCl3): δ 2.40 (s, 3H), 4.86 (s, 1H), 6.82 (m, 1H), 7.06 (t, 1H), 1H), 7.45 (m, 1H), 7.60 (m, 1H), 7.88 (s, 1H), 8.80 (s, 1H) ppm. 7.15-7.40 (m, 6H) ppm. IR (neat): 3609, 3365, 3115, 3039, IR (Nujol): 3314, 3141, 1699, 1630, 1607, 1577 cm-1. 2919, 1709, 1611, 1578, 1475 cm-1. 3′-Hydroxybiphenyl-3-sulfonic Acid Amide (3r).38 A 3′-Aminobiphenyl-3-ol (3j). A light-brown powder was pale yellow oil was obtained. Yield: 86% (3.323 g). MS (EI): obtained. Yield: 56% (0.363 g). Mp: 154-159 °C (acetone/ + 1 + 1 m/z 249 (M ), 169 (100). H NMR [CD3C(O)CD3]: δ 6.65 (br s, petroleum ether). MS (EI): m/z 185 (M ), 63 (100). H NMR 2H), 6.90 (dd, 1H), 7.15 (m, 2H), 7.30 (t, 1H), 7.60 (t, 1H), 7.85 (CDCl3): δ 3.77 (br s, 2H), 6.58 (m, 1H), 6.73-7.20 (m, 7H), (m, 1H), 8.12 (m, 1H), 8.60 (br s, 1H) ppm. IR (Nujol): 3360, 8.59 (s, 1H) ppm. IR (neat): 3381, 3305, 1586 cm-1. 3100, 2933, 2855, 1693, 1599 cm-1. 3′-Fluorobiphenyl-3-ol (3k).35 A yellow oil was obtained. 1-(3′-Hydroxybiphenyl-3-yl)ethanone (3s). A yellowish- Yield: 97% (0.639 g). MS (EI): m/z 188 (M+, 100), 159. 1H NMR gray oil was obtained. Yield: 69% (2.270 g). MS (EI): m/z 212 (CDCl3): δ 5.74 (s, 1H), 6.89 (m, 1H), 7.01-7.45 (m, 7H) ppm. + 1 -1 (M ), 197 (100). H NMR (CDCl3): δ 2.68 (s, 3H), 5.18 (s, 1H), IR (neat): 3354, 3066, 2925, 2854, 1611, 1578 cm . 6.88 (m, 1H), 7.12 (t, 1H), 7.20 (d, 1H), 7.35 (t, 1H), 7.54 (t, 3′-Phenoxybiphenyl-3-ol (3m). A light-brown oil was 1H), 7.79 (d, 1H), 7.95 (d, 1H), 8.18 (t, 1H) ppm. IR (Nujol): obtained. Yield: 95% (0.872 g). MS (EI): m/z 262 (M+), 77 -1 1 3348, 1660, 1616, 1600, 1584 cm . (100). H NMR (CDCl3): δ 4.81 (s, 1H), 6.80-7.44 (m, 13H) 3′-Hydroxybiphenyl-3-carbonitrile (3t).39 White crystals ppm. IR (film): 3381, 3061, 3033, 2925, 2849, 1703, 1600, 1573 were obtained (lit.: liquid).39 Yield: 90% (2.723 g). Mp: 107- cm-1. 108 °C (petroleum ether). MS (EI): m/z 195 (M+), 140 (100). [1,1′;3,1′′]-Terphenyl-3-ol (3n). Gray crystals were ob- 1H NMR and IR spectra are according to literature.39 tained. Yield: 97% (0.836 g). Mp: 93-95 °C (cyclohexane). MS 40 + 1 Biphenyl-3,3′-diol (3u). A yellow solid was obtained. (EI): m/z 246 (M , 100). H NMR (CDCl3): δ 4.97 (s, 1H), 6.83- Yield: 89% (2.569 g). MS (EI): m/z 186 (M+, 100). 1H NMR 7.81 (m, 13H) ppm. IR (KBr): 3470, 3391, 3046 cm-1. (CDCl3): δ 4.97 (br s, 2H), 6.84 (ddd, 2H), 7.05 (m, 2H), 7.16 3′-Propylbiphenyl-3-ol (3p). A colorless oil was obtained. (ddd, 2H), 7.32 (t, 2H) ppm. IR (neat): 3465, 3270, 2922, 2852, Yield: 65% (0.483 g). MS (EI): m/z 212 (M+, 100). 1606, 1577, 1433, 1181 cm-1. General Procedure for the Synthesis of 3′-(or 4′-)- 3′-Hydroxymethylbiphenyl-3-ol (3v). A pale green oil Substituted Biphenyl-3-ols (3c,o,q). To a stirred, cooled (0 + was obtained. Yield: 92% (2.855 g). MS (EI): m/z 200 (M ), °C), 1 M solution of BBr3 in CH2Cl2 (7.8 mL) was added a 1 153 (100). H NMR (CDCl3): δ 1.83 (br s, 1H), 4.78 (s, 2H), solution of the opportune methoxybenzene 8c,o,q (3 mmol) in 5.12 (s, 1H), 6.84 (m, 1H), 7.07 (m, 1H), 7.18 (d, 1H), 7.28- dry CH2Cl2 (35 mL) under N2 atmosphere. The mixture was 7.53 (m, 4H), 7.59 (s, 1H) ppm. IR (neat): 3338, 2930, 2881, stirred at room temperature (1 h for 3q;5hfor3c;18hfor -1 1703, 1595, 1578 cm . 3o), then quenched with 2N Na2CO3, and extracted with 3′-(2-Hydroxyethyl)biphenyl-3-ol (3w). A pale yellow oil EtOAc. The combined organic phases were dried (Na2SO4) and was obtained. Yield: 63% (2.092 g). MS (EI): m/z 214 (M+), concentrated. Purification of the residue by column chroma- 1 183 (100). H NMR [CD3C(O)CD3]: δ 2.09 (s, 1H), 2.89 (t, 2H), tography (cyclohexane/EtOAc 8:2 for 3c,o; 7:3 for 3q) gave 3c,q 3.80 (t, 2H), 6.83 (m, 1H), 7.09-7.50 (m, 7H), 8.44 (s, 1H) ppm. as solids and 3o as an oil. IR (neat): 3327, 1600, 1578 cm-1. 3′-Hydroxybiphenyl-4-carboxylic Acid Amide (3c). White General Procedure for the Synthesis of 3′-(or 4′-)- crystals were obtained. Yield: 76% (0.486 g). Mp: 187-189 + Substituted Biphenyl-3-ols (3a,b,d,e,g,h,j,k,m,n,p). The °C (EtOAc/petroleum ether). MS (EI): m/z 213 (M ), 139 (100). 1 opportune methoxybenzene 8a,b,d,e,g,h,j,k,m,n,p (3.5 mmol) H NMR [CD3C(O)CD3]: δ 6.70 (br s, 1H), 6.87 (m, 1H), 7.17 and 57% HI (4.375 mL) were refluxed with vigorous stirring (m, 2H), 7.30 (m, 1H), 7.54 (br s, 1H), 7.70 (d, 2H), 8.01 (d, (2 h for 3a,b,d,h,j,k,n;3hfor3e,m,p; 7.5 h for 3g). The 2H), 8.62 (br s, 1H) ppm. IR (KBr): 3449, 3339, 3291, 3209, -1 mixture was cooled, diluted with H2O, neutralized with 2 N 1652, 1610, 1413, 1299 cm . ′ NaHCO3, and extracted with EtOAc (CH2Cl2 in the case of 3 -Benzylbiphenyl-3-ol (3o). An amber oil was obtained. + 1 3b,h,n). The combined organic phases were dried (Na2SO4) and Yield: 75% (0.458 g). MS (EI): m/z 260 (M , 100). H NMR concentrated. Purification of the residue by column chroma- (CDCl3): δ 4.05 (s, 2H), 6.80 (dd, 1H), 7.10-7.50 (m, 12H) ppm. tography (cyclohexane/EtOAc 9:1 for 3a; 9:1 to 8:2 for 3b,h,n,p; IR (KBr): 3376, 3028, 2923 cm-1. 8:2 for 3e,k,m; 7:3 for 3g; 6:4 for 3j; 1:1 for 3d) gave 3′-Nitrobiphenyl-3-ol (3q).43 Yellow crystals were ob- 3a,b,d,e,g,j,n as solids and 3h,m,h,k,p as oils. tained. Yield: 90% (0.581 g). Mp: 118-119 °C (EtOAc/ 5006 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 21 Mor et al. petroleum ether) [lit.: 117 °C (benzene)].43 MS (EI): m/z 215 Synthesis of Cyclohexylcarbamic Acid 3′-Aminometh- + 1 (M , 100), 168. H NMR (CDCl3): δ 4.95 (s, 1H), 6.92 (m, 1H), ylbiphenyl-3-yl Ester (4z). To a stirred solution of 4t (0.175 7.12 (t, 1H), 7.23 (d, 1H), 7.38 (t, 1H), 7.62 (t, 1H), 7.91 (d, g, 0.54 mmol) in THF (5 mL) was added Raney nickel. The 1H), 8.22 (d, 1H), 8.45 (t, 1H) ppm. IR (Nujol): 3500, 3093, mixture was hydrogenated at 4 atm at 65 °C for 3 h, cooled, 1605, 1535, 1508, 1344 cm-1. filtered on Celite (DANGER! Raney nickel is pyrophoric if Synthesis of (3′-Hydroxybiphenyl)carbamic Acid Ben- allowed to dry), and concentrated. Purification of the residue zyl Esters (3x,y). To a stirred solution of NaHCO3 (0.157 g, by column chromatography (cyclohexane/EtOAc 6:4) and re- 1.87 mmol) in H2O (3 mL) were added the opportune biphenyl- crystallization gave 4z as white crystals. Yield: 30% (0.053 3-ol 3d,j (0.315 g, 1.7 mmol) and benzyl chloroformate (0.290 g). Mp: 154-157 °C (with decomposition) (EtOAc/petroleum + g, 1.7 mmol, 0.24 mL). The mixture was stirred 16 h, and then ether) (sealed capillary tube). MS (EI): m/z 324 (M ), 82 (100). 1 - a further amount of benzyl chloroformate (one drop) was H NMR [CD3C(O)CD3]: δ 1.04 1.89 (m, 10H), 3.55 (m, 1H), added. The mixture was stirred for other 4 h and then 4.38 (d, 2H), 5.55 (br d, 1H), 5.97 (br s, 1H), 6.83 (m, 1H), 7.10- extracted with EtOAc. The combined organic layers were dried 7.55 (m, 7H), 8.64 (s, 1H) ppm. IR (Nujol): 3365, 3338, 1599, -1 44 over Na2SO4 and evaporated. Purification of the residue by 1573 cm . Anal. (C20H24N2O2)C,H,N. column chromatography (cyclohexane/EtOAc 7:3) gave 3x as Synthesis of 3-Bromobenzoic Acid Amide (6i). To a an oil and 3y as a solid. stirred solution of 3-bromobenzonitrile (3.555 g, 19.5 mmol) ‚ (3′-Hydroxybiphenyl-3-yl)carbamic Acid Benzyl Ester in dioxane (74 mL) were added NaBO3 4H2O (8.269 g, 53.74 (3x). A white oil was obtained. Yield: 94% (0.510 g). MS (EI): mmol) and H2O (74 mL). The mixture was stirred at 80 °C for + 1 16 h, cooled, H O was added and the mixture was extracted m/z 319 (M ), 91 (100). H NMR (CDCl3): δ 4.72 (s, 1H), 5.23 2 (s, 2H), 6.81-7.62 (m, 14H) ppm. IR (Nujol): 3386, 3321, 1709, with CH2Cl2. The combined organic layers were dried over Na2- 1611, 1584, 1546 cm-1. SO4 and evaporated. Purification of the residue by column chromatography (hexane/EtOAc 2:8) and recrystallization gave (3′-Hydroxybiphenyl-4-yl)carbamic Acid Benzyl Ester 6i as colorless tablets. Yield: 80% (3.12 g). Mp: 156-157 °C (3y). White crystals were obtained. Mp: 175-176 °C (EtOAc/ (EtOH) (lit. 156 °C).45 MS (EI): m/z 199 (M+); 183 (100). petroleum ether). Yield: 66% (0.358 g). MS (EI): m/z 319 (M+), 1 (b) Pharmacology. Membrane fractions were prepared 91 (100). H NMR (CDCl3): δ 4.93 (s, 1H), 5.23 (s, 2H), 6.73 (br s, 1H), 6.80 (m, 1H), 7.04 (t, 1H), 7.14 (m, 1H), 7.30-7.59 from brain homogenates, and FAAH activity was assayed 3 3 (m, 10H) ppm. IR (Nujol): 3381, 1675, 1599, 1538 cm-1. using [ H]anandamide (anandamide[ethanolamine- H], Ameri- can Radiolabeled Chemicals, ARC, St. Louis, MO, 60 Ci/mmol) Synthesis of Cyclohexylcarbamic Acid Aminobiphen- as a substrate. Rat brain membranes (50 µg protein) were yl-3-yl Esters (4d,j). To a stirred solution of the convenient incubated for 30 min at 37 °C in buffer containing [3H]- carbamic acid biphenyl ester 4x,y (0.356 g, 0.8 mmol) in THF anandamide and varying concentrations of test compounds. (15 mL) were added MeOH (0.26 mL) and 10% Pd/C (0.039 g). At the end of the incubation period, we stopped the reactions The mixture was hydrogenated at 3 atm at 65 °C for 24 h, with a mixture of chloroform/methanol and measured [3H]- cooled, filtered on Celite, and concentrated. Purification of the ethanolamine in the aqueous phase by liquid scintillation residue by column chromatography (cyclohexane/EtOAc 6:4) counting.19,20 [3H]Anandamide transport assays were con- gave 4d,j as solids. ducted in human astrocytoma cells, preincubating cells with ′ Cyclohexylcarbamic Acid 4 -Aminobiphenyl-3-yl Ester inhibitors for 10 min at 37 °C, prior to exposure to [3H]- (4d). An off-white powder was obtained. Yield: 42% (0.106 7 anandamide for 4 min. CB1 and CB2 binding assays were g). Mp: 121-125 °C (acetone/petroleum ether). MS (EI): m/z conducted in rat cerebellar membranes (27000g) and CB2- + 1 - 310 (M ), 185 (100). H NMR (CDCl3): δ 1.20 2.05 (m, 10H), overexpressing CHO cells (purchased from Receptor Biology- 2.80 (m, 2H), 3.60 (m, 1H), 4.97 (br d, 1H), 6.74 (m, 2H), 7.05 Perkin-Elmer, Wellesley, MA), respectively, using [3H]WIN- - (m, 1H), 7.29 7.42 (m, 5H) ppm. IR (Nujol): 3457, 3403, 3294, 55212-2 (NEN-Dupont, Boston, MA, 40-60 Ci/mmol, 10 nM) -1 ‚ 1747, 1709, 1627, 1605 cm . Anal. [C19H22N2O2 0.10MeC(O)- as a ligand.8,46 Cholinesterase assays were conducted with a Me] C, H, N. commercial kit (Sigma, St. Louis, MO), using purified enzymes Cyclohexylcarbamic Acid 3′-Aminobiphenyl-3-yl Ester (electric eel acetylcholinesterase type V-S and horse serum (4j). Light yellow crystals were obtained. Yield: 83% (0.206 cholinesterase, both from Sigma, St. Louis, MO) and following g). Mp: 129-134 °C (EtOH). MS (EI): m/z 310 (M+), 185 (100). vendor’s instructions. 1 - H NMR (CDCl3): δ 1.22 2.06 (m, 10H); 3.59 (m, 1H); 3.75 (c) QSAR. MRA calculations were performed with an Excel (m, 2H); 4.96 (br d, 1H); 6.68 (m, 1H); 6.90-7.41 (m, 7H) ppm. (Microsoft Co., version 97) spreadsheet, employing the built- IR (Nujol): 3466, 3388, 3297, 1740, 1707, 1609, 1581, 1540 in statistical functions and automated macro procedures. -1 cm . Anal. (C19H22N2O2)C,H,N. Substituent properties were parametrized by the variables π, 2 Synthesis of 3-Methoxy-3′-propenylbiphenyl (11). To π σm, F, R, MR, L, B1, B5. When available, their values were a stirred solution of ethyltriphenylphosphonium bromide (10) taken from the van de Waterbeemd data set;47 for the benzyl, (4.529, 12.2 mmol) in dry THF (122 mL) was added 2M n-BuLi hydroxyethyl, and aminomethyl substituents they were taken 48 2M in hexane (6.1 mL) under N2 atmosphere at 0 °C. The from the Hansch collection; π and MR values for the mixture was stirred for 2 h and cooled (-78 °C), and then 9 cyclohexylcarbamoyloxy group (4l) were estimated by the (2.589 g, 12.2 mmol) was added. The mixture was allowed to Chem Prop module in Chem Draw,49 and those of the electronic reach room temperature, refluxed for 20 h, and cooled. H2O variables from similar alkylaminocarbonyloxy groups in the was cautiously added, and the mixture was extracted with Hansch collection. MRA models were calculated for all the EtOAc. The combined organic layers were dried (Na2SO4) and possible combinations of maximum five variables. Standard evaporated. Purification of the residue by column chromatog- deviation of the errors in prediction (SDEP) and the relative raphy (cyclohexane/EtOAc 9:1) gave 11 as a colorless oil. predictivity parameter, q2, were calculated by cross-validation, Yield: 41% (1.122 g). MS (EI): m/z 224 (M+, 100), 181, 165, omitting one compound at a time from the set, according to 115. the leave-one-out technique (LOO).50 Synthesis of 3-Methoxy-3′-propylbiphenyl (8p). 11 (d) Molecular Modeling. Molecular models were built, (1.009 g, 4.8 mmol) was dissolved in EtOH (60 mL) and, after refined, and analyzed by Sybyl version 6.8.51 Energy calcula- addition of PdO2 (0.103 g, 0.843 mmol), hydrogenated at room tions were performed employing the Merck molecular force temperature at 3 atm for 3 h. The mixture was filtered on field (MMFF94s for geometry optimization and MMFF94 for Celite and concentrated. Purification of the residue by column molecular dynamics),52 implemented in Sybyl, with the dielec- chromatography (cyclohexane/EtOAc 95:5) gave 8p as a yellow tric set constant to 1. FAAH structure coordinates were taken oil. Yield: 76% (0.826 g). MS (EI): m/z 226 (M+), 197 (100). from those of the covalent adduct with a MAP chain, reported 1 53 H NMR (CDCl3): δ 0.98 (t, 3H), 1.70 (m, 2H), 2.66 (t, 2H), in the Protein Data Bank (PDB code: 1MT5). The first 3.88 (s, 3H), 6.91 (m, 1H), 7.14-7.41 (m, 7H) ppm. IR (neat): structure refinement (addition of missing side chains and 3060, 3029, 2990, 2959, 2920, 2870, 2833, 1596, 1573 cm-1. hydrogens) was done by the Biopolymer module, and it was Fatty Acid Amide Hydrolase Inhibitors Journal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5007 followed by a visual inspection of histidine tautomerism, which (14) Cravatt, B. J.; Lichtman, A. H. Fatty acid amide hydrolase: an was modified to maximize the number of possible hydrogen emerging therapeutic target in the endocannabinoid system. - bonds. The geometry of added atoms was then relaxed by Curr. Opin. Chem. Biol. 2003, 7, 469 475. ‚ (15) Bisogno, T.; De Petrocellis, L.; Di Marzo, V. Fatty Acid Amide energy minimization to a gradient of 0.1 kcal/(mol Å), the MAP Hydrolase, an Enzyme with Many Bioactive Substrates. Possible atoms removed, and the hydrogen atoms at catalytic site Therapeutic Implications. Curr. Pharm. Des. 2002, 8, 533-547. residues were reassigned to get a hydrogen bond between (16) Yarnell, A. Soothing Pain the Natural Way. Chem. Eng. News Ser241 and Ser217, and one between Ser217 and the NH2 2002, 80, 32. group of Lys142. The inhibitors were then fitted into the (17) Smith, A. FAAH better anxiolytics? Nat. Rev. Drug Discovery enzyme channel, optimizing their position and conformation 2003, 2, 92. first by the Dock_minimize procedure, then by energy mini- (18) Wendeler, M.; Kolter, T. Inhibitors of Endocannabinoid Degra- dation: Potential Therapeutics for Neurological Disorders. An- mization of the complex, allowing movements of the residues gew. Chem., Int. Ed. 2003, 42, 2938-2941. at maximum 8 Å from the inhibitor. Molecular dynamics (19) Kathuria, S.; Gaetani, S.; Fegley, D.; Valin˜ o, F.; Duranti, A.; simulations (step size of 1 fs) started with seven 3000-step Tontini, A.; Mor, M.; Tarzia, G.; La Rana, G.; Calignano, A.; heating cycles, gradually raising T from 0 to 310 K and Giustino, A.; Tattoli, M.; Palmery, M.; Cuomo, V.; Piomelli, D. continued with 200000 fs of simulation; after 100000 fs of Modulation of Anxiety Through Blockade of Anandamide Hy- equilibration, a snapshot of the trajectory was saved every 500 drolysis. Nat. Med. 2003, 9,76-81. fs for subsequent analysis. The surface of the enzyme channel (20) Tarzia, G.; Duranti, A.; Tontini, A.; Piersanti, G.; Mor, M.; Rivara, S.; Plazzi, P. V.; Park, C.; Kathuria, S.; Piomelli, D. shown in Figures 2 and 4 was built by the MOLCAD module Design, Synthesis, and Structure-Activity Relationship of Alkyl- in Sybyl. carbamic Acid Aryl Esters, a New Class of Fatty Acid Amide Hydrolase Inhibitors. J. Med. Chem. 2003, 46, 2352-2360. Acknowledgment. This work was supported by (21) Reggio, P. H.; Traore, H. Conformational requirements for MIUR (Ministero dell’Istruzione, dell’Universita` e della endocannabinoid interaction with the cannabinoid receptors, the Ricerca), Universities of Parma and Urbino, and the anandamide transporter and fatty acid amidohydrolase. Chem. Phys. Lipids 2000, 108,15-35. National Institute on Drug Abuse (to D.P.). The CCE (22) Barnett-Norris, J.; Hurst, D. P.; Lynch, D. L.; Guarnieri, F.; (Centro di Calcolo Elettronico) and CIM (Centro Inter- Makriyannis, A.; Reggio, P. H. Conformational Memories and facolta` Misure) of the University of Parma are gratefully the Endocannabinoid Binding Site at the Cannabinoid CB1 Receptor. J. Med. Chem. 2002, 45, 3649-3659. acknowledged for supplying the Sybyl software license. (23) Bracey, M. H.; Hanson, M. A.; Masuda, K. R.; Stevens, R. C.; Cravatt, B. F. 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